JPS60197690A - Salt of dc-52 - Google Patents

Abstract

PURPOSE:To provide the titled substance having improved freeze-drying stability and storage stability, by adding a specific inorganic acid or organic acid to DC- 52. CONSTITUTION:The compound of formula (DC-52) produced by Streptomyces melanovinaceus DO-52 (FERM-P No.5911) is added with 0.5-2.0 equivalent, preferably 1 equivalent of an inorganic acid (e.g. hydrochloric acid, phosphoric acid, etc.), a sulfonic acid (e.g. methanesulfonic acid), an acidic amino acid (e.g. glutamic acid), citric acid, trans-aconitic acid, alpha-ketoglutaric acid, itaconic acid, malonic acid or ascorbic acid to obtain the objective substance. EFFECT:The salt has antitumor activity against the leukemia P-388 ascites tumor cell.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to salts of DC-52. More specifically, the present invention uses DC-52 and 0.5-2.0 equivalents of inorganic acids, sulfonic acids, and acidic amino acids relative to DC-52.

The present invention relates to pharmacologically acceptable DC,52 acid addition salts with citric acid, trans-aconitic acid, α-ketoglutaric acid, itaconic acid, malonic acid or ascorbic acid.

DC-52 is Streptomyces melanobinaceus (
Streptomyces melanovinace
us) A substance produced by DO-52 (Feikoken Bibori No. 5911), which has the following structure and is similar to Leukemia P-388.
It has antitumor activity against 111 tree tumor cells (Japanese Patent Application Laid-Open No. 170189/1989).

However, DC-52 is unstable and decomposes during purification and powdering (for example, during freeze-drying), making it impossible to obtain highly pure DC-52, and decomposition also progresses during storage.

When we measured the stability of DC-52 against p) in an aqueous solution, as shown in Table 1, the stability improved slightly at <pH 5 to 6, but when 2 equivalents and equivalents of DC-52 It has been found that when hydrochloric acid is added to lower the pH to below pH II, the stability is extremely reduced.

*Equivalent ratio to IDC-52. In addition, DC-5
For 2, 1 mole was defined as 2 equivalents.

*2 This is the value immediately after preparation.

Analysis of DC=52 is 1000μg/m as DC-52
10μ of each solution adjusted to I) was added to nucleotidyl 10C1
8 (manufactured by Gascro Industries) as a filler and acetonitrile:
0. IM phosphate buffer (pH 7,0) -1!
This was performed by subjecting to high performance liquid chromatography (measurement wavelength: 271 nm) using If as a mobile phase. .

Therefore, the stability of DC-52 hydrochloride was worse than that of I) C-52, and it was thought that it would be difficult to improve the stability by using a salt of DC-52. However, other than that, DC.

When the aqueous hydrochloride solution of No. 52 was immediately freeze-dried to powder and its purity was measured, it was found that powder with extremely high purity could be obtained as shown in Table 2.

*Equivalent ratio to IDC-52*2 Analysis of DC-52 was performed by subjecting each powder to high performance liquid chromatography under the above conditions noted in Table 1.

*3 pH when the powder is made into an aqueous solution of 1000 μg/ml In other words, the stability during freeze-drying is significantly improved for hydrochloride.

Therefore, we investigated salts with other acids and found that there were differences in stability depending on the type of acid, and that among those that had good stability during freeze-drying, there were many that had good stability during storage. Ta. It has also been found that, as in the case of hydrochloride, stability is affected by the equivalent ratio of DC-52 to acid in the salt.

(The present invention is based on the fact that specific acid addition salts of DC-52 have different stability in aqueous solution and stability during powdering and storage, and that DC-52 in acid addition salts and acids This is based on various new findings such as the fact that stability varies depending on the equivalent ratio of , and does not simply relate to acid addition salts.

The antitumor activity of various salts of DC-52 of the present invention is
It has an activity equivalent to that of

Among the acids used in the present invention, inorganic acids include hydrochloric acid, hydrobromic acid,
Hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Sulfonic acids include methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, methanedisulfonic acid.

α,β-ethanedisulfonic acid, benzenesulfonic acid, p
-) luenesulfonic acid, etc., and acidic amino acids include glutamic acid, aspartic acid, etc., respectively.

The ratio of acid to DC-52 in the acid addition salt of DC-52 of the present invention is 0.5-2.0 times equivalent (however, DC-52
, 1 mole = 2 equivalents) is necessary in order to maintain a high degree of stability, and approximately 1.0 times the equivalent is particularly preferred.

Further, the acid addition salt of the present invention is preferably in a powder state. The powdering method is not particularly limited, but freeze-drying is preferred.

The acid addition salt of DC-52 of the present invention can be prepared by reacting DC-52 with a certain amount of an appropriate acid in an aqueous solution using a conventional method. I contraction・
It can be obtained by drying or freeze-drying. After the reaction, purification operations such as recrystallization may be added.

Note that DC-52 can be obtained by the method described in Japanese Patent Application Laid-open No. 170189/1989. That is, glucose, fructose, sucrose,
Starch, etc., ammonium sulfate, ammonium chloride, urea as nitrogen sources.

Hefton, meat extract, yeast extract, corn steep liquor, soybean flour, etc., inorganic salts such as salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium phosphate, manganese sulfate, zinc sulfate, etc., and other vitamins are appropriately combined. Inoculate the culture medium and perform liquid culture. Culture solution temperature is 25-40°C.

When culture is usually carried out for 1 to 7 days at pH 4 to 10, DC-52 accumulates in the culture solution and in the bacterial cells. The bacterial cells are separated from the cultured solution, and the culture solution is passed through a porous resin. Thereafter, the adsorbed substance is eluted using methanol, acetone, ethyl acetate, or the like. This eluate was concentrated, adsorbed onto Celite powder,
DC,52 is eluted with methanol, acetone, etc., and further purified by a combination of solvent extraction, partition chromatography, etc.

Next, examples of the present invention will be shown.

Example 1゜ Streptomyces milanovinaceus DO as inoculum
-52, the strain was cultured in a 21-volume Erlenmeyer flask on type 1 medium [K(14% 1M SO, 71(,00,5g/
j! , KII2P0. 1.5g/I1. Ammonium sulfate 5.0 g/β, sucrose 20 g/β
.

Fructose lOg/1. Glucose 10g/l,
Corn steeple force - 5,0 g / j! , CaC
O520g/β, pH 7.0] Inoculated into 300ml,
Culture with shaking (22 rpm) at 30°C for 48 hours. 200ml of the 1st type culture solution submerged in this way
Type 2 medium in a 1 volume fermenter (same ingredients as type 1 medium) 1004! In addition, culture was carried out at 30°C for 48 hours with aeration (rotation number 15'Orpm, aeration rate 70F/min). The obtained second type culture solution 100 I/liter was added to the fermentation medium [glucose 50 g/A, Kll] in a two-layer fermenter.
□PO40.3g/β, -HPO40.4g/'
l.

MgSO4・7H200, 2g/l, powdered human bean meal 20g
/R, CaCL 1 g/ji', p) (7,0] 1
In addition to fart, aerated agitation culture at 30℃ for 72 hours [rotation speed 15
0rpm.

Ventilation amount 200j! / min: l. After completion of the culture, 50 pg/ml of DC-52 is accumulated in the culture solution.

A filter aid is added to this culture solution and the bacterial cells are filtered out to obtain a culture filtrate of 1.1%. The culture filtrate is passed through a porous adsorption resin (Diaion) IP-10 (Mitsubishi Chemical Industries KK brand name) 50p to adsorb DC-52 to the resin. After adsorption, wash with water 6
Elute with % acetone water, concentrate the eluate under reduced pressure to obtain a concentrated solution of 10%
get f. Ethanol 4H is added to this concentrate to precipitate impurities, and the mixture is centrifuged to obtain a supernatant. This supernatant liquid is concentrated under reduced pressure, and a solution of 15 liters of ethanol:water is added thereto to form a charge liquid for silica gel.

Silica gel packed with ethanol:water-15=1 eluent (Kanto Chemical Department, 100-100-2O0+>10
When the charge liquid is passed through J2 and then developed with an eluent, DC, -52 is eluted in an amount between 4 and 6 times the amount of resin. After collecting the fraction containing this DC-52 and concentrating it under reduced pressure,
Add water and concentrate again to remove ethanol.

The concentrated liquid was filtered with a millibore filter (pore size 0.45 microns) to remove cloudy substances, and the DC-52 concentration was 42.
250 ml of g/R filtrate are obtained. 5 ml of this filtrate
After cooling to ℃, hydrochloric acid was added to adjust the pH of the solution to 2.8, and freeze-drying was performed to obtain DC-52 hydrochloride powder [equivalent ratio: 0.90 (equivalent ratio of hydrochloric acid to DC-52) )]5508m is obtained.

Melting point: 160-180°C Ultraviolet absorption spectrum: Fig. 1 Infrared absorption spectrum (Br tablet method) Fig. 2 Example 2゜DC-52 ml J bore 10 m of filtrate obtained in Example 1
1 was cooled to 5°C, phosphoric acid was added to adjust the pH to 2.7,
Then, freeze-drying is performed to obtain 524 mg of DC-52 phosphate powder (equivalent ratio: 0.90).

Melting point: 128-132°C Ultraviolet absorption spectrum: Fig. 7 Infrared absorption spectrum (KBr tablet method): Fig. 8 Example 3゜DC-52mi IJ pore filtrate 10+ obtained in Example 1
The mixture was cooled to 5° C., 224 mg of ascorbic acid was added, and then freeze-dried to obtain 705 mg of DC-52 scorbate powder (equivalence ratio 0.50).

Melting point: 129-132°C Ultraviolet absorption spectrum: Fig. 3 Infrared absorption spectrum (KBr tablet method): Fig. 4 Example 4゜ DC-52 mm IJ bore filtrate 10 m obtained in Example 1
1 was cooled to 5°C and citric acid was added to bring the solution to pH 3.
0, and then freeze-dried to obtain 630 mg of DC-52 citrate powder (equivalent ratio: 1.1).

Melting point: 105-150°C Ultraviolet absorption spectrum: Fig. 5 Infrared absorption spectrum (Br tablet method): Fig. 6 Example 5゜ Each flavor of DC-52 obtained according to Examples 1 to 4 i# Immediately after preparation, Shichinosai (r) (1:1 equivalent ratio of Ni52 and acid) (lyophilized product) was capped under vacuum in an ampoule at 30°C.
High performance liquid chromatography after storage in a thermostatic chamber for 60 days [Filling agent: Nucleozil 10C18, developing solvent Niacetonitrile: 0.1 M phosphate buffer (p) 17
．． 0)=1:9] The results of the analysis are shown in Table 3.

It is clear from Table 3 that the acid addition salts of the present invention have excellent stability during the powdering process and generally excellent stability after storage.

Table 3: pH when the powder immediately after production is made into an aqueous solution of 1.000 μg/ml Example 6゜ DC-52 citrate with various equivalent ratios was prepared according to Examples 1 to 4, and Example 5 As well as purity.

The residual rate after storage at pH 1 was examined. The results are shown in Table 4.

Table 4 Example 7. Antitumor Activity A cell suspension of 5×10 6 cells/m+ of Leukemia P-388 cells, which have been passaged intraperitoneally in DBA/2 male mice, is prepared in sterile physiological saline. 0.2 m of this cell suspension
CDF weighing approximately 25 g is intraperitoneally transplanted into a group of 5 male mice. DC-
52, DC-52 hydrochloride and DC-52 citrate are administered intraperitoneally at the respective doses shown in the table.

Table 5 shows the results of antitumor activity expressed as T/C% (percentage of the average survival days (T) of the drug-administered group to the average survival days (C) of the non-drug-administered group).

From the upper table of Table 5, it can be said that the antitumor activities of the three compounds are almost the same.

[Brief explanation of drawings]

Figure 1.3, 5.7 fiDC-52 hydrochloride, DC-52
Ascorbate, DC-52 Citrate, DC-52
! The ultraviolet absorption spectrum of I phosphate is shown. Figure 2.4.6.8 shows DC-52 hydrochloric acid m, DC-52 ascorbate, DC-52 citrate, DC-521
The infrared absorption spectrum of J-phosphate is shown.

Claims (1)

[Claims] [1) A compound represented by the formula (hereinafter referred to as DC-52) and DC-
52 to 0.5-2.0 equivalents of inorganic acid. Sulfonic acid, acidic amino acid, citric acid, trans-
Pharmacologically acceptable DC- with aconitic acid, α-ketoglutaric acid, itaconic acid, malonic acid or ascorbic acid
52 acid addition salt. (2) Inorganic acids are hydrochloric acid, hydrobromic acid, and hydroiodic acid. Sulfuric acid, nitric acid or phosphoric acid, the sulfonic acid is methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, methanedisulfonic acid, α,β-ethanedisulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid, and the acidic amino acid The acid addition salt according to claim 1, wherein is glutamic acid or aspartic acid. (3) The amount of acid used relative to D(, -52 is 0.5-2.
3. The acid addition salt according to claim 1 or 2, which has an equivalent of 0. (4) The acid addition salt according to claim 3, wherein the amount of acid used is 1 equivalent relative to DC-52. (5) The acid addition salt according to claim 1 or 2, wherein the acid addition salt is in a powder state. (6) The acid addition salt according to claim 5, wherein the acid addition salt is a lyophilized product.